Generally, the inventive technology disclosed herein relates to methods and apparatus for reducing protein content in sperm cell extenders. More specifically, this inventive technology may involve eliminating the protein content of a cryoprotectant component used in a multiple step sperm cell extension process. The inventive technology may be particularly suited for use in sorted sperm applications.
Sperm cell extenders may be commonly used in a variety of biological disciplines that require working with sperm cells. For example, one discipline that may make extensive use of sperm cell extenders is the field of artificial insemination. Whereas natural insemination may involve direct male to female insemination, artificial insemination may typically involve collecting sperm cells from a male, performing a degree of human manipulation of such sperm cells removed from their natural environment, and then inserting the manipulated sperm cells into a female. The precise degree of human manipulation may vary depending on the precise nature of the particular application. For example, some human manipulation may simply involve dividing a collected sperm sample into multiple doses for use in multiple insemination events, possibly with multiple female animals. However, other applications may require more intensive human manipulation.
For example, human manipulation in some applications may involve sorting sperm cells into populations based on characteristics exhibited by sperm cells. One such application may include the use of flow cytometery to separate sperm cells into populations of X-chromosome-bearing and Y-chromosome bearing sperm cells. A flow cytometer may typically accomplish such separation by flowing sperm cells entrained in a fluid stream one at a time through an interrogation region, where information about each sperm cell may be obtained. Interrogation may typically be accomplished through the use of optics, for example perhaps by intersecting a laser beam with a sperm cell and measuring the resulting light scatter or fluorescence. The determination of a sex characteristic perhaps may be made by staining the sperm cells with a fluorescent dye that binds to the DNA within individual sperm cells. When a laser illuminates individual sperm cells, the dye may fluoresce. Sorting of sperm cells according to a sex characteristic may then be accomplished, perhaps by recognizing that sperm cells bearing an X chromosome have more DNA than sperm cells bearing a Y chromosome, thus possibly emitting more fluorescent light when excited by a laser and perhaps allowing the cell to be identified and separated.
Another example of human manipulation may involve perhaps freezing sperm cells for use at a later time. Freezing sperm cells may often be critical to the effective use of sperm cells, because freezing may preserve at least some degree of the viability of sperm cells for a period of time extended beyond a point at which such viability otherwise may typically become compromised. Such extension of sperm cell viability may be accomplished in freezing techniques perhaps by slowing the metabolism of sperm cells and perhaps extending their useful life accordingly. In particular, it perhaps may be known that sperm cell metabolism may be slowed by about 50% approximately for every 10 degrees Celsius to which a sperm cell is cooled. Moreover, frozen sperm cells may be packaged in formats convenient for particular applications, for example perhaps as frozen straws, frozen pellets, or other forms of frozen artificial samples. Frozen sperm cells also may lend themselves well to transportation over large distances, for example as where a sperm cell collection facility, sperm cell extension facility, and artificial insemination facility may be widely dispersed at different locations.
It may be appreciated that the removal of sperm from their natural environment may remove them from natural support mechanisms that maintain their viability. Sperm cell extenders may act to restore at least a degree of such support to sperm cells. For example, one function of sperm cell extenders may be to buffer sperm cells, for example perhaps by adjusting the pH or osmoality of a medium into which sperm cells may be placed. Another function of sperm cells extenders perhaps may be to provide nutrients to sperm cells or to serve as a sperm cell energy source. In freezing applications, a further function may be to serve as a cryoprotectant to minimize the adverse effects of freezing upon sperm cells. It may be appreciated that such functions of sperm cell extenders may be accomplished at least to some degree by the constituent parts that make up any individual sperm cell extender.
For example, protein content may be a frequently used constituent part of various types of sperm cells extenders. Protein content may serve one or more functions in a sperm cell extender. A primary purpose of protein content may be to provide nutrients and perhaps serve as an energy source for sperm cells. However, some kinds of protein may also have a cryoprotectant function, for example, perhaps the use of lipoproteins to replace lipids lost from sperm cell membranes that may be due to a freezing process. Moreover, protein content in sperm cell extenders may take a variety of forms. Some protein content may be plant-based, for example lecithin derived from soy. Other protein content may be animal-based, for example, perhaps egg yolk derived from sources including common hen's eggs.
Cryoprotectants also may be an example of a frequently used constituent part of various types of sperm cell extenders. Moreover, cryoprotectants may take a variety of forms in sperm cell extenders. One commonly used cryoprotectant may be glycerol. Glycerol may protect sperm cells during a freezing process, perhaps by binding to water contained within and surrounding a sperm cell, perhaps dehydrating the sperm cell as a result, and accordingly perhaps reducing the formation of intracellular ice that may cause damage to the sperm cell. However, using glycerol to cryoprotect sperm cells also may entail certain disadvantages. For example, glycerol may pose at least a degree of toxicity to sperm cells, the effect of which may become more pronounced with larger amounts of glycerol. Further, glycerol may be hyperosmotic to sperm cells, which may result in a degree of shock to sperm cells to which glycerol has been added. In particular, such hyperosmotic properties of glycerol may cause a sperm cell coming into contact with glycerol to rapidly shrink or expand as a result of a difference in solute concentration across the sperm cell's membrane. Such rapid shrinking and expanding may perhaps cause damage to a sperm cell.
Accordingly, certain procedures may have been developed for sperm cell extenders to minimize the adverse effects of glycerol on sperm cells. For example, as a practical matter it may perhaps be recognized that combining glycerol with sperm cells at reduced temperatures may reduce the toxic effects of glycerol on sperm cells. Accordingly, sperm cell extenders using glycerol often may be prepared in a multiple step process involving two or more extender fractions. More particularly, certain sperm cell extenders may contain an “A” fraction without glycerol and a “B” fraction with glycerol. This may allow a sperm cell extender to be prepared in two or more steps, for example, a first step in which sperm cells may be added to the A fraction of a sperm cell extender at perhaps room temperature, followed by a second step in which the sperm cells added to the A fraction are cooled to a lower temperature, and the B fraction containing glycerol added at such a lower temperature. Moreover, to mitigate the hyperosmotic effects of glycerol on sperm cells, the B fraction perhaps may be added in multiple steps, possibly so as to reduce the shock to sperm cells by subjecting sperm cells to lowered amounts of glycerol at each added glycerol step. The number of steps in which glycerol may be added may vary from perhaps as few as two steps or four steps to perhaps a great number of steps, including perhaps adding glycerol drip-wise over a period of time.
However, the interaction of glycerol with other sperm cell extender components in such procedures may entail significant drawbacks. In particular, protein components of sperm cell extenders such as egg yolk may pose complications for the handling of such extenders when present in the B fraction. This may be due to the volumetric bulk that such protein components create in a sperm cell extender. This phenomenon perhaps may be highlighted by the use of egg yolk in the B fraction of a sperm cell extender requiring centrifugation. Centrifugation may be a commonly used technique in various sperm cell applications to concentrate sperm cells. For example, in flow cytometery applications, the passage of sperm cells through a flow cytometer may tend to dilute the concentration of sperm cells to a lower concentration than that found in nature. This may be because flow cytometers typically may require entraining sperm cells in a sheath fluid, which may add to the volume of material in which sperm cells are contained. Centrifugation may return sperm cells to a higher concentration perhaps by subjecting them to centrifugal forces and concentrating them accordingly. However, centrifuging the B fraction of a sperm cell extender containing egg yolk may be problematic because the volumetric bulk of lipoproteins contained in the egg yolk may tend to compact any sperm cells that may be present in the B fraction, perhaps with the result of crushing or otherwise damaging such sperm cells.
As a result, it may perhaps be necessary to clarify the B fraction of a sperm cell extender containing protein content such as egg yolk. The goal of clarification may be to confer a lower and more uniform degree of density to such a protein-containing extender, perhaps in particular by removing clumps or other locally dense regions due perhaps to protein concentrations such as lipoprotein components of egg yolk, so that centrifugation perhaps may be accomplished without adversely compacting sperm cells. Clarification may be accomplished by any of various suitable methods, for example perhaps by filtration. However, all forms of clarification may require a dedication of resources to accomplish. For example, clarification may entail material costs such as filters or other required devices, labor costs which may tie up personnel resources that otherwise could be dedicated elsewhere, time costs which may slow down a sperm cell extension process, and financial costs related to all of the foregoing.
Moreover, simply preparing a B fraction of a sperm cell extender to contain protein content such as egg yolk may entail a degree of inherent drawbacks. Similarly to clarification, preparation of such a B fraction may entail material costs, labor costs, time costs, and financial costs. Moreover, the tendency toward spoliation over time due to the protein content of such a B fraction may further complicate its use. In particular, because such a B fraction may not keep well, it may require preparation on an as-needed basis, perhaps disrupting schedules and reducing efficiencies that could be realized if the B fraction otherwise could be prepared in large quantities ahead of time. This drawback may be particularly acute in situations where a sperm cell application may require a relatively high ratio of B fraction to A fraction. The spoliation tendencies of such a B fraction may also pose a contamination risk, for example as where the B fraction may perhaps become contaminated with bacteria due to spoliation, which may adversely affect a sperm cell application in which the contaminated B fraction inadvertently may be used. Such spoliation tendencies also may limit the use of such a B fraction in situations where environmental conditions cannot be closely monitored, for example as where it may be desired to transport the B fraction from one location to another perhaps over a large distance.
The foregoing problems regarding conventional sperm cell extenders may represent a long-felt need for an effective solution to the same. While implementing elements may have been available, actual attempts to meet this need may have been lacking to some degree. This may have been due to a failure of those having ordinary skill in the art to fully appreciate or understand the nature of the problems and challenges involved. As a result of this lack of understanding, attempts to meet these long-felt needs may have failed to effectively solve one or more of the problems or challenges here identified. These attempts may even have led away from the technical directions taken by the present inventive technology and may even result in the achievements of the present inventive technology being considered to some degree an unexpected result of the approach taken by some in the field.